Process for the purification of amylases

ABSTRACT

The present procedure concerns a process for the purification of beta-amylases comprising treating a crude beta-amylase solution derived from wheat bran, soy bean or fresh sweet potato with an adsorbent such as bentonite, acidic clay, kaolinite and activated clay at a pH lower than 5.0 to adsorb the beta-amylases, washing out the adsorbent with water, and then eluting the adsorbed betaamylases with a solution with an ionic strength over 0.5 Mu and a pH exceeding 5.0.

United States Patent [1 1 Masuda [111 3,769,168 Oct. 30, 1973 PROCESS FOR THE PURIFICATION OF AMYLASES [75] Inventor:

[73] Assignee: Hayashibara Company, Okayama,

Japan 221 Filed: June 11,1971

21 Appl.No.: 15 2,41 5 7 Kazuo Masuda, Okayama, Japan [30] Foreign Application Priority Data OTHER PUBLICATIONS Dixon et al., Enzymes p. 41-43, published by Academic Press, 2nd ed., 1964.

McLaren, A. D. J. Phys. Chem. Vol. 58, p. 129-137,

Primary Examiner-Lionel M. Shapiro [5 7 ABSTRACT The present procedure concerns a-process for the pu- 4 rification of beta-amylases comprising treating a crude beta-amylase solution derived from wheat bran, soy bean or fresh sweet potato with an adsorbent such as bentonite, acidic clay, kaolinite and activated clay at a pH lower than 5.0 to adsorb the beta-amylases, washing out the adsorbent with water, and then eluting the adsorbed beta-amylases with a solution with an ionic strength over 0.5 and a-pH exceeding 5.0.

6 Claims, No Drawings PROCESS FOR THE PURIFICATION OF AMYLASES DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a process characteristic in the purification of enzymes, comprising treatment of crude beta-amylase solution derived from wheat bran, soy beans or sweet potato with adsorbents, such as kaolinite, activated clay, acidic clay, bentonite, and the like, to adsorb the enzymes, separation of the enzymes and subsequent elution of the separated enzymes.

The production of pure beta-amylase is of an indispensable importance for the industrial production of pure maltose or maltose syrups. However, a process for the production of pure beta-amylase is hardly known except those for experimental applications. Known sources for betaamylases are primarily of botanical sources which include wheat bran, soy beans, sweet potato, etc.

The only reported processes for the extraction and purification of beta-amylases derived from botanical The invention will now be described in detail.

The enzymatic sources used were rasped (extract) solution of raw sweet potato, wheat bran extract solution and solution extracted from soy bean flour defatted at low temperature with water. Such extract solutions do not contain other varieties of amylases and thus are advantageous enzymatic sources for industrial practice. In order to study the pHs of the solutions to be employed in adsorption and elution, determination of the pH stabilities were carried out. As regards betaamylases of sweet potato the residual activities were determined after allowing standing 3 U/ml of enzymatic solution (containing M/20 of acetate buffer solutions of various pHs at -l5C for 16-20(hours). At pH 3 the enzyme solutions were inactivated completely whereas no inactivation was observed at pH 4-11, es-

sources are (l) the process described by Th. Sabalitschka, and R. Weidlich Biochem. Z. 211 229-38(1929), (2) Oskar Holmbergh; Arkivkemi, Mineral Geol. 11B. No. 6pp(l932), and (3) Arnoldk. Balls. Mayo, K. Walden, U.S. Pat. No. 2,496,261, Feb. 7, 1950. According to the process (1) the beta-amylase is absorbed from a malt extract solution using kao-linite at pH 4.5 and then eluted with a phosphate buffer (pH 7:6). However the process results in an enzyme recovery rate of slightly over 50 percent. In (2) is described a process comprising preparation of a 50 percent alcohol solution with an enzyme extract solution derived from barley malt, absorption and separation of the betaamylases using starch, and washing in order to separate the alpha-amylases from the resultant product. The

process described in (3) comprises treating of the beta- The inventors investigated various methods applicable for industrial use for the absorption and purification of crude enzyme solutions extracted from enzymatic sources abundant with enzymes, such as sweet potatoes, soy beans and wheat bran, using adsorbents. The

adsorbents investigated in the detailed studies on the conditions of adsorption and elution were kaolinite or clays of activated clay and acidic clay. These investigations resulted in the findings that purification of en'- zymes was attainable with a recovery rate of nearly 100 percent by adsorbing the enzymes at lower than pH 5.0 and then eluting the resultant product using a buffer with a pH higher than 5.0 and an ionic strength of over tablishing the fact that enzymes were stable within such pH range.

The following results were obtained by comparisons of the adsorption amount at pH 5.0 and the elution amount with water. The results are shown in Table I.

TABLE I. Adsorption of amylases derived from sweet potato Adsor- Units of amylases The amount of enzymes bent present in the adeluted with water sorbed amount per Wash liquor gram adsorbent I 11 Ill Bentonite 4860 0 0 0 Acidic clay 4860 0 0 0 Kaolinite 4310 30 O Activated clay 4850 1570 30 0 At pH 5.0 each amount of adsorption was extremely large, while on the other hand there was hardly any elution with water.

The results of comparison tests on the amounts of elution at different pHs are listed in Table II.

TABLE II. Variations of Elutions Depending on Variations of pHs Amount of enzymes Amount of enzymes eluted with acidic Na,CO;,NaHCO buffer, pH 9-l2 As obvious from the above listed results elution at lower than pH 5.0 is difficult and the maximum amount of elution is achieved within the pH range of 7.0-9.0. With amylases extracted from wheat bran and soy beans it was found that the enzymes could be adsorbed safely and eluted at a pH of 5.0-9.0.

The ionic strengths of the elution solutions were determined according to the following procedures. To enzyme solutions with 102 units per ml and pH 3.8 were added 2 percent of kaolinite, adsorbed, washed with water thrice and then the amounts of elution of the enzymes were compared at ionic strengths of 0.27, 0.8, and 2.4 with the use of 20 m1 of buffer (pl-l 4.0-11.0). The results obtained were as follows.

TABLE 111. Variations of Elution at Respective Ionic Strengths pl-l Elution p.=0.27 Elution p.=0.8 Elution p.=2.4

Elution Total amount of enzymes eluted,

Total Units l 1.0 l 12 21 l 144 As the above results show elution was not sufficient at ionic strength of 0.27 and the maximum elution rate was attained at the pH 7.0-10.0. The fact that the results corresponded with those of amylases derived from soy beans and wheat bran were also established. Adsorption of the enzymes by adsorbents .is generally faster at lower temperature, while elution is faster at higher temperature. However it is preferable that adsorption and elution be carried out at lower than room temperature because of a wider range of pH stability of enzymes resulting at lower temperature.

The methods of adsorption and elution based on the above conditions will be further described.

1. Adsorption and elution of bea-amylases derived from sweet potato.

100 ml of filtered solution extracted from sweet p0 tato was admixed with O, 0.5, 1, 1.5 and 2 percent (by volume) of adsorbents, stirred at 30C for 30 minutes, adsorbed and filtered, and the enzymatic activities of the filtrates were determined, designating the loss of activity as the adsorbed enzymatic activities. Elution was carried out with 100 ml of M/S Na CO -,NaHCO buffer, ionic strength of p.=0.8.standing at 1 0-15C for hours, and then the eluted amounts of enzymes was determined. Since when the enzymes present in enzyme solutions are 100% adsorbed the amount of adsorption per minimum amount of adsorbent exhibits its v maximum and the elution rate alsodisplays its maximum, the amounts of enzymatic adsorption and elution, and elution rates obtained by 100 percent adsorption are listed in the following table.

TABLE IV. Adsorption and elution of amylases derived from sweet potato Adsorbent Adsorption: Elution: A B C D E Bentonite 0 7,650 0 0.5 0 15,300 6,990 45.7 Acidic clay 0 8,880 0 2 1,200 3,840 2,797 72.2 Kaolinite 0 9,170 O I 2.5 0 3,610 3,640 100 Activated clay 0 6,980 0 I 1.5 110 4,580 4,763 104 Notes:

A; The amount of adsorbent added, (by volume) B; Enzymes present in filtered solution, U/100 rnl C; The amount of enzymes adsorbed U/gram adsorbent D; The amount of enzymes eluted, U/gram adsorbent E; Elution rate,

Similarly aqueous extract solutions of wheat bran and soy beans extracted at low temperature were filtered, adsorbed and eluted, the results of which are given .in the following. tables.

TABLE V. Adsorption and elution of wheat bran beta-amylases Adsorbent Adsorption: Elution A B C D E Bentonite 0 8 ,350 0 8 40 1 .040 83 7 80.4 Acidic clay 0 l .760 0 10 0 176 1 33 .3 Kaolinite 0 1,760 0 8 20 21 7 l 96 90.5 Activated clay 0 8,350 0 8 40 1 ,040 980 94. 1 Notes:

A; The amount of adsorbent added, (by volume) B; Enzymes present in filtered solution U/100 ml C; The amount of enzymes adsorbed Ulgram adsorbent D; The amount of enzymeseluted, U/gram adsorbent I E; Elution rate,

TABLE V1. Adsorption and elution of soy bean beta-amylases Adsorbent Adsorption Elution:

A B C D E Bentonite 0 3,990 0 2 60 1,970 596 33.5 Acidic clay 0 3,990 0 6 33 660 381 57.6 Kaolinite 0 1,950 0 l0 '0 1,950 1,302 66.9 Activated clay 0 3,990 0 6 20 661 242 36.1 Notes:

A; The The amount of adsorbent added, (by volume) B; Enzymes present in filtered solution U/ 100 m1 C; The amount of enzymes adsorbed U/gram adsorbent D; The amount of enzymes eluted, Ulgram adsorbent E; Elution rate,

As the above results show when beta-amylases derived from sweet potato are adsorbed with adsorbents, especially with kaolinite or activated clay, the amount of enzyme adsorbed are several times of that obtained from soy bean or wheat bran beta-amylases and since the amount of elute is 100 percent the process according to the present invention is feasible and advanta geous for the industrial purification of enzymes. In the case of soy bean and wheat bran amylases the amount adsorbed is relatively small and displays a slightly lower tendency .of elution rate. However, although the amount adsorbed in the case of wheat bran amylases is small the process is applicable on an industrial scale. The results of determinations of the activities (specific activities) per mg nitrogen of the thus obtained enzymes are listed in the following table.

TABLE V'II.-OOMPARISON OF SPECIFIC ACTIVITIES WITH SWEET POTATO BETA-AMYLASES As is obvious from the above table the specific activities of enzyme solutions purified by adsorption and elution were increased by two to six times compared with that of the enzyme solution used as the starting material and also the amount of nitrogen impurities were decreased to one sixth. Especially in the case when kaolinite is employed a substantial contribution of the process can be expected, showing an enzymatic yield of nearly 100 percent. 1

As described in details above, a process suitable for the adsorption and purification of beta-amylases present in wheat malt with using kaolinite has been reported. However, the yield of the process is 50-60 percent, which are not conditions normally used on an industrial scale. On the other hand no detailed information is available on processes for beta-amylases derived from sweet potato or wheat bran. The process investigated by the inventors is applicable for enzymes derived from sweet potato and wheat bran which are sources of relatively pure beta-amylases. The inventors succeeded in the development of a process which is characterized in that the recovery rate of enzymes is nearly 100 percent and that high purity beta-amylases are obtainable with high yields and which comprises the complete adsorption of the enzymes at pH lower than 5.0 using adsorbents, such as kaolinite and activated clay and then completely the enzymes by extraction at a pH greater than 5.0, preferably at a pH range of 7.0-9.0 with water of an ionic strength abovep THE METHOD OF THE DETERMINATION OF ENZYMATIC ACTIVITIES A reaction solution comprising 5 ml of 1 percent starch solution, 4 ml of M/lO acetate buffer solution (pH 5.0 and 1 ml of enzyme solution is incubated at 40C for 30 minutes, inactivated with the addition of 2 ml of alkali solution described in the Fehling-Lehmann- Schoorl method, and then with the addition of 2 ml of cupric nitrate the formed reduced sugars are determined quantitatively. The activity of beta-amylase that effected the formation of mg of glucose by a 30 minutes reaction at 40C from the formed amount of reduced sugars obtained by the subtraction of blanks of substrates and enzymes was disignated as one unit per one ml. The employed enzyme solutions were those diluted in the range in which the formed amounts of reduced sugars were in proportion to the enzymatic activities. Further details of the invention are illustrated by the'following examples which represent preferred embodiments. All proportions given are by weight unless otherwise indicated.

EXAMPLE 1 To 4 ml of filtered and washed liquor of rasped sweet potato, which exhibited a beta-amylase activity of 91.7 u/ml (total activity 3.67 X 10 units), was added 100g of kaolinite and stirred at 30C for 30 minutes and then filtered. No enzymatic activity was observed in the filtered liquor on determination. To the filter cake was added 300 ml of M/5 Na CO -,NaI-ICO buffer (pH 9.0), allowed to standing at C for 16 hours, filtered with suction, and then the filter cake was mixed again with 300 ml of said buffer and filtered with suction. The thus obtained filter cake was suspended again in 100 ml of said buffer and filtered. Enzymatic activity was noticed as being substantially absent in the cake. The enzyme solutions obtained by extracting the filter cakes thrice by the above treatment were put together to give an enzyme solution which was converted into a neutralized solution of one liter with the addition of acetate and then the enzymatic activity was determined, resulting in a total enzymatic activity of 3.45 X 10 units. The recovery rate and specific activity were 94 percent and u/mg N=62 respectively.

The enzyme solution was added to a liquefied starch solution of sweet potato at the rate of two units per gram starch. The mixture was incubated at pH 6.0 and 45C for 10 hours and thus a maltose solution which possessed a maltose content of 5.3 percent was obtained. Further 20 units of isoamlyase were added per gram starch, respectively, and the mixture was incubated at 45C and pH 5.5 for 40 hours. Thus a yield of a maltose solution with a purity of 93 percent was possible.

EXAMPLE 2 To 100 ml of an aqueous extraction solution of wheat bran (enzymatic activity 350units per ml) was added 35 mg of activated clay. The mixture was stirred at pH 4.0 at 30C for 30 minutes, filtered with suction, washed with 50 ml of water twice, 200 ml of a M/5 Na CO -NaI-ICO buffer (p.=0.8) were added, and allowed to stand at 510C for 20 hours. Following subsequent filtering out of the activated clay the resultant product was extracted again with 100 ml of said buffer, and thus 94% of the enzymatic activity was recovered. Said aqueous extraction solution of wheat bran was treated similarly using kaolinite as the adsorbent. In this case percent of the enzymatic activity was recovered. Since the specific activity of the enzyme was 66 units per mg N, showing an increase of 78 times compared with theoriginal solution, and a pure betaamylase in which no other types of amylases were present was obtained, said enzymes possess a purity which is sufficient for industrial applications.

What we claim is:

1. A process for the purification of beta-amylases comprising treating a crude beta-amylase solution derived from wheat bran, soy bean or fresh sweet potato with an adsorbent selected from the group consisting of bentonite, acidic clay, kaolinite and activated clay with' a pH lower than 5.0 to adsorb said beta-amylases, washing the adsorbent enzyme complex with water to remove the impurities therein, and then eluting the adsorbed beta-amylases from the adsorbent-enzyme complex with a solution with an ionic strength over 0.5g. and a pH exceeding 5.0.

2. A process according to claim 1 wherein said adsorption and elution are carried out at lower than 30C.

3. A process according to claim 1 wherein said enzymes are adsorbed with an absorbent of the minimum amount that effects percent adsorption of said enzymes on said adsorbent, and in that the elution is carried out following said adsorption.

4. A process according to claim 1 wherein an enzyme solution obtained by the extraction of fresh sweet potato is adsorbed with kaolinite or activated clay to obtain substantially pure enzymes at a higher yield.

5. A process according to claim 1 wherein enzymes derived from wheat bran are recovered at a higher yield using kaolinite or activated clay.

6. A process according to claim 1 wherein enzymes derived from soy bean are recovered at an especially high yield by adsorption with kaolinite and elution.

i it I k 

2. A process according to claim 1 wherein said adsorption and elution are carried out at lower than 30*C.
 3. A process according to claim 1 wherein said enzymes are adsorbed with an absorbent of the minimum amount that effects 100 percent adsorption of said enzymes on said adsorbent, and in that the elution is carried out following said adsorption.
 4. A process according to claim 1 wherein an enzyme solution obtained by the extraction of fresh sweet potato is adsorbed with kaolinite or activated clay to obtain substantially pure enzymes at a higher yield.
 5. A process according to claim 1 wherein enzymes derived from wheat bran are recovered at a higher yield using kaolinite or activated clay.
 6. A process according to claim 1 wherein enzymes derived from soy bean are recovered at an especially high yield by adsorption with kaolinite and elution. 